Growth factors derived from a human malignant glioma cell line,U-251MG

Summary A human malignant glioma cell line, U-251 Mg, cultured under serum free conditions, was shown to produce a growth factor for BALB/c 3T3 cells (glioma-derived growth factor-1, GDGF-1). The biological activity of GDGF-1 resided in a heat- and acid-resistant protein with a molecular weight (MW) of 25 kDa estimated by gel permeation chromatography. GDGF-1 activity was neutralized by a goat anti-human platelet derived growth factor (PDGF) antibody, indicating that the two factors were immunologically related. Furthermore, U-251 Mg cells constitutively expressed c-sis mRNA. When U-251 Mg cells were stimulated with bacterial lipopolysaccharide, 2 novel growth factors (GDGF-2 and GDGF-3) were produced in addition to the PDGF-like substance. GDGF-2 was determined to be >100 kDa MW and was not neutralized by the goat anti-PDGF antiserum. The biological activity of GDGF-3 was also heat- and acid- resistant with an apparent 14 kDa MW This factor also did not show any common antigenicity with PDGF. GDGF-2 and GDGF-3 are currently under investigation and evidence as to their natures will be published elsewhere. Our findings with this glioma cell line provide further evidence that inappropriate expression of growth factor-related genes could play important autocrine role(s) in the processes leading to malignant transformation and/or uncontrolled proliferation and may provide a paracrine stimulus for such processes as glioma neovascularization.     

ADAR1 mRNA在肝癌及癌旁组织的表达及意义

目的探讨ADAR1mRNA在肝细胞肝癌(HCC)及癌旁组织的表达及意义。方法采用半定量逆转录-聚合酶链反应(RT-PCR)分别检测诊断为HCC41例患者的肝癌及癌旁组织RNA编辑酶ADAR1mRNA的表达。结果所有肝癌组织RNA编辑酶ADAR1均有表达,相对表达量为3.340±0.863;所有癌旁组织有RNA编辑酶ADAR1表达,相对表达量为0.801±0.209;对照的26例正常肝组织及其他肝脏疾病标本RNA编辑酶ADAR1的相对表达量为0.880±0.226。将肝癌组织和癌旁组织的ADAR1mRNA表达量进行成组t检验,两者间存在显著性差异(t=18.30,P<0.001);肝癌组织和对照肝组织的ADAR1mRNA表达量进行成组t检验,两者间亦存在显著性差异(t=17.65,P<0.001);将癌旁组织和对照肝组织检测到的ADAR1mRNA的表达量进行成组t检验,两者间差异无统计学意义(t=1.64,P>0.10)。将从同一患者取材的肝癌组织和癌旁组织的ADAR1mRNA表达量进行配对t检验,两者间存在显著性差异(t=18.53,P<0.001)。结论ADAR1mRNA在肝癌组织的表达增高,可能在肝癌的发生机制中起一定的作用。     

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.     

Over-expression of APAF-1 and caspase-9 augments radiation-induced apoptosis in U-373MG glioma cells.

The p53 tumor-suppressor gene plays a critical role in radiation-induced apoptosis. Several genes, including Bax and Fas, are involved in p53-mediated apoptosis, and their over-expression enhances the degree of radiation-induced apoptosis. Apaf-1 and caspase-9 have been reported to be downstream components of p53-mediated apoptosis, suggesting that these genes play a role in radiation-induced apoptosis. In this study, we transduced U-373MG cells harboring mutant p53 with the Apaf-1 and/or caspase-9 genes via adenoviral (Adv) vectors concomitant with X-ray irradiation and evaluated the degree of apoptosis. The percentage of apoptotic cells in U-373MG cells co-infected with the Adv for Apaf-1 (Adv-APAF-1) and that for caspase-9 (Adv-Casp9) and treated with irradiation (24%) was much higher than that in cells co-infected with Adv-APAF-1 and Adv-Casp9 and not treated with irradiation (0.86%) and that in cells infected with either Adv-APAF-1 or Adv-Casp9 and treated with irradiation (2.0% or 2.6%, respectively). The apoptosis induced by co-transduction of Apaf-1 and caspase-9 and irradiation was repressed in cells that were co-infected with the Adv for Bcl-X(L) but not in cells co-infected with the Adv for Bcl-2. These results indicate that Apaf-1 and caspase-9 play a role in radiation-induced apoptosis in cancer cells harboring mutant p53. Bcl-X(L) may be critically involved in the radioresistance of cancer cells by repressing Apaf-1- and caspase-9-mediated apoptosis. Expression of Apaf-1 and caspase-9 in tumors may be an important determinant of the therapeutic effect of irradiation in cancer treatment.     

RNA interference against SPARC promotes the growth of U-87MG human malignant glioma cells

Malignant glioma is a highly invasive brain tumor resistant to conventional therapies. Secreted protein acidic and rich in cysteine (SPARC) has been shown to facilitate glioma invasion. However, the effects of SPARC on cell growth have yet to be adequately elucidated. In this study, we constructed a plasmid expressing shRNA against SPARC, evaluated the effect of SPARCshRNA on SPARC expression and then assessed its effect on cell growth in U-87MG cells. Using plasmid-delivered shRNA, we effectively suppressed SPARC expression in U-87MG cells. Cell growth curves and colony formation assay suggested that the introduction of SPARCshRNA resulted in an increase of cell growth and colony formation. We also showed that knockdown of SPARC expression was capable of promoting the cell cycle progression from the G1 to S?phase. However, no difference was found in the level of apoptosis. A molecular analysis of signal mediators indicated that the inhibition of p-c-Raf?(Ser259) and accumulation of p-GSK-3β?(Ser9) and p-AKT?(Ser473) may be connected with the growth promotion by SPARC?shRNA. Our study may provide an insight into the biological function of SPARC in glioma.     

EZH2基因沉默对人脑胶质瘤U251细胞增殖和凋亡影响的研究

目的:研究RNA干扰技术抑制zeste基因增强子人类同源物2(EZH2)的表达对人胶质瘤U251细胞增殖及凋亡的影响.方法:构建靶向EZH2基因的shRNA质粒并转染至U251细胞中,采用RT-PCR和蛋白质印迹法检测EZH2 mRNA和蛋白的表达情况,利用四甲基偶氮哇盐(MTT)实验和Annexin V-FITC/PI流式细胞术实验观察转染后U251细胞增殖和凋亡情况.结果:靶向EZH2基因的shRNA质粒成功抑制了U251细胞EZH2基因的表达,mRNA和蛋白表达抑制率分别为56.00％和88.73％;转染shRNA EZH2后,U251细胞的生长受到明显抑制(P＜0.01),在转染96 h后,生长抑制率达35.79％;转染48 h后,U251细胞早、晚期凋亡率分别为(26.59±0.83)％和(38.63±0.80)％,较阴性质粒组和空白对照组均增加,以晚期明显,差异有统计学意义,P＜0.01.结论:EZH2基因的沉默能有效抑制胶质瘤U251细胞的增殖、促进其凋亡,提示EZH2可能成为胶质瘤基因治疗的新靶点.     

Effect of interferon-ß on the cell cycle of human glioma cell line U-251 MG: flow cytometric two-dimensional (BrdU/DNA) analysis

Summary This paper describes the determination of the effect of IFN-ß on U-251 MG cells using the bromodeoxyuridine (BrdU/DNA) analysis technique. The cell cycle perturbation of exponentially growing cells was estimated by a newly developed two-dimensional analysis of sequential BrdU/DNA distributions measured at 4-hr intervals after IFN-ß administration. The U-251 MG cell line was sensitive to IFN-ß, and cell proliferation was inhibited by 50.0% at 48 hr. Analysis of DNA histograms indicated that IFN-ß, accumulated the cells in the S-phase, from 16 to 48 hr after treatment. In the two-dimensional analysis, labeled cells treated with IFN-ß moved from the S-phase through the G₂M-phase and then entered the G₁-phase within 12 hr after the initial treatment, in a pattern similar to labeled cells untreated with IFN-ß. After 16 hr, labeled cells treated with IFN-ß, began to accumulate in the S-phase and remained there even after 48 hr. These results imply that IFN-ß may have an effect on the G₁-phase, thereby inducing S-phase accumulation of human glioma cell line U-251 MG.     

Forced expression of Sox21 inhibits Sox2 and induces apoptosis in human glioma cells

Numerous studies support a role for Sox2 to keep stem cells and progenitor cells in an immature and proliferative state. Coexpression of Sox2 and GFAP has been found in regions of the adult brain where neural stem cells are present and in human glioma cells. In our study, we have investigated the roles of Sox2 and its counteracting partner Sox21 in human glioma cells. We show for the first time that Sox21 is expressed in both primary glioblastoma and in human glioma cell lines. We found that coexpression of Sox2, GFAP and Sox21 was mutually exclusive with expression of fibronectin. Our result suggests that glioma consists of at least two different cell populations: Sox2⁺/GFAP⁺/Sox21⁺/FN^? and Sox2^?/GFAP^?/Sox21^?/FN⁺. Reduction of Sox2 expression by using siRNA against Sox2 or by overexpressing Sox21 using a tetracycline‐regulated expression system (Tet‐on) caused decreased GFAP expression and a reduction in cell number due to induction of apoptosis. We suggest that Sox21 can negatively regulate Sox2 in glioma. Our findings imply that Sox2 and Sox21 may be interesting targets for the development of novel glioma therapy.     

微小染色体维持蛋白2在脑胶质瘤中的表达

目的探讨微小染色体维持蛋白2(MCM2)在脑胶质细胞瘤中表达的意义及与Ki67的关系。方法应用免疫组织化学SABC法检测35例胶质瘤组织和5例正常脑组织中MCM2和Ki67的表达情况。结果MCM2和Ki67在正常脑组织中不表达,胶质瘤中MCM2和Ki67的表达随肿瘤病理级别的增加而增加,MCM2和Ki67的表达呈明显正相关(r=0.832,P<0.001)。而MCM2要比Ki67高,尤其在Ⅰ~Ⅱ级之间(PMCM2=0.004,PKi67=0.502)和Ⅲ~Ⅳ级之间(PMCM2=0.002,PKi67=0.039)。MCM2和Ki67的标记指数在生存时间≥2年组和生存时间<2年组差异均具有显著性(P=0.001;P=0.032),胶质瘤中MCM2和Ki67的表达在不同性别、年龄间的比较,差异均无显著性(P>0.05)。结论MCM2比Ki67在脑胶质瘤中能更好地反映细胞的增殖情况,并对预后判断更有指导意义。     

Leptin促进人脑胶质瘤U87MG细胞的迁移和侵袭

目的:探讨瘦素(leptin)对人脑胶质瘤U87MG细胞迁移及侵袭能力的影响及其机制。方法:Leptin处理U87MG细胞,采用细胞划痕实验检测U87MG细胞的迁移能力,Transwell实验检测U87MG细胞的侵袭能力,RT-PCR及Western blot-ting法检测U87MG细胞中MMP-2及MMP-9 mRNA和蛋白的表达。结果:Leptin明显促进U87MG细胞迁移能力[(152.42±3.29)vs(83.24±2.61)μm,P<0.05]和侵袭能力[(31.78±5.04)vs(17.03±2.41)个细胞,P<0.05],leptin能显著上调U87MG细胞中MMP-2、MMP-9 mRNA[(0.76±0.04)vs(0.35±0.02),(0.84±0.02)vs(0.41±0.06);均P<0.05]及蛋白[(0.79±0.03)vs(0.23±0.01),(0.81±0.05)vs(0.39±0.03);均P<0.05]的表达。MMP抑制剂GM6001(10μmol/ml)可以逆转leptin对U87MG细胞迁移[(82.05±2.98)vs(81.76±3.25)μm,P>0.05]和侵袭能力[(19.23±2.46)vs(18.02±1.98)个细胞,P>0.05]的影响。结论:Leptin可以促进人脑胶质瘤U87MG细胞的侵袭及迁移,其机制可能与上调MMP-2、MMP-9的表达有关。     

The mechanism of polyamine analog-induced enhancement of cisplatin cytotoxicity in the U-251 MG human malignant glioma cell line

Purpose: During the last decade, several polyamine analogs have been developed as antineoplastic agents that replace intracellular polyamines but cannot mimic the biological functions of polyamines related to cell growth. It has been shown that pretreatment of several human brain tumor cell lines with some of these polyamine analogs increases the cytotoxicity of cis-diamminedichloroplatinum (CDDP). It has also been established that some of these polyamine analogs affect chromatin organization. In the study reported here we attempted to elucidate the mechanism by which polyamine analog-induced changes in DNA and chromatin structure may increase CDDP cytotoxicity. Methods: We studied the micrococcal nuclease sensitivity of the nuclei and measured the amount of platinum incorporated into the nucleosomal and linker regions of chromatin isolated from CDDP-treated U-251 MG human malignant brain tumor cells with or without pretreatment with two cytotoxic polyamine analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-diazanonadecane (BE-4-4-4-4). Results: The pretreatment with the polyamine analogs decreased the MNase sensitivity and increased the incorporation of CDDP preferentially into the linker region of the chromatin. Conclusions: Pretreatment of cells with polyamine analogs probably alters the structure and/or the organization of the linker region such that more CDDP incorporates into the linker DNA. This is probably the reason for the observed enhancement of CDDP cytotoxicity in the polyamine analog-pretreated cells. Received: 10 March 1997 / Accepted: 14 July 1997     

Deletion of the RNA‐editing enzyme ADAR1 causes regression of established chronic myelogenous leukemia in mice

Patients with chronic myelogenous leukemia (CML) respond well to tyrosine kinase inhibitors (TKIs) of the Bcr‐Abl oncoprotein. However, intolerance and resistance to these agents remains a challenge, and TKIs are unable to eradicate rare leukemia‐initiating cells. Leukemia treatment would benefit from a better understanding of molecular signals that are necessary for the survival of leukemia‐initiating cells but dispensable for normal hematopoietic stem cells. Leukemia‐initiating cells in CML can arise from myeloid progenitor cells, a population that we have reported in normal hematopoiesis to depend on the RNA‐editing enzyme adenosine deaminase acting on RNA‐1 (ADAR1). We now report that Bcr‐Abl transformed leukemic cells were ADAR1‐dependent in a conditional ADAR1 knockout mouse model. ADAR1 deletion reversed leukocytosis and splenomegaly, and preferentially depleted primitive Lin‐Sca+Kit+ (LSK) leukemic cells but not LSK cells lacking the leukemic oncoprotein. ADAR1 deletion ultimately normalized the peripheral white blood count, eliminating leukemic cells as assessed by PCR. These results uncover a novel requirement for ADAR1 in myeloid leukemic cells and indicate that ADAR1 may comprise a new molecular target for CML‐directed therapeutics.     

Antiproliferative properties of the lazaroids U- 83836E and U- 74389G on glioma cells in vitro

The 21-aminosteroids (lazaroids) are a new family of steroid compounds that inhibit lipid peroxidation reactions. They are novel antioxidant agents, which have been shown to have antiproliferative properties on cancer cells and also are thought to prevent free radical-mediated blood-brain barrier damage. In order to understand the effect of lazaroids on glioma, we tested U-83836E and U-74389G at doses ranging between 0.1 100 m mM on primary cultures of glioblastoma multiforme from three patients, rat C6 glioma cell line, and 5 th subculture established from one of the patients. The effects of both compounds on cell proliferation were determined using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. U-83836E in the primary cultures was found to have 50% inhibitory concentrations (IC50 ) of 6.30, 6.75 and 6.50 m mM, respectively. The IC50 value of U-74389G was calculated as 91 m mM in only one of the patients. On C6 glioma cells, while the IC50 of U-83836E was 45 m mM, U-74389G showed no cytotoxic effect. On the 5 th subculture, U-83836E had an IC50 of 37.5 m mM, but the cytotoxic effects of U-74389G was less than in that of the primary culture. In conclusion, these compounds were found to be more cytotoxic in primary culture than the cell lines and there were also differences between their members in the inhibition of cell survival.     

羟基脲对人脑胶质瘤U251细胞放化疗增敏作用的研究

目的:探讨羟基脲(HU)联合替莫唑胺(TMZ)加放疗(RT)对人脑胶质瘤U251细胞放化疗(CRT)敏感性的影响。方法:体外培养U251细胞,采用CCK8实验检测不同浓度HU、TMZ及不同条件处理后细胞的增殖能力;流式细胞术检测细胞凋亡及细胞周期分布情况;Transwell小室、划痕实验评估细胞侵袭、迁移能力变化;We...     

Human interleukin-2 activated cytotoxic cells kill autologous glioma cells in vitro

Summary We have cultured peripheral blood lymphocytes (PBL) from glioblastoma patients in recombinant interleukin-2 (IL-2) containing medium for a period of 5 days. The cytotoxicity of these cells was tested on ⁵¹Cr-labelled autologous dissociated glioblastoma cells which had not been cultured. Significant cytotoxicity against glioma cells was observed in seven out of nine cases. IL-2 activated PBL from normal donors were equally cytotoxic against these glioma cells. Autologous lymphocytes activated by phytohaemagglutinin were also lysed in most cases, and the erythroleukemia cell line K562 was highly susceptible to the cytotoxic capability of the IL-2 activated PBL. In cold target inhibition experiments, K562 inhibited the cytotoxicity against both autologous and allogenic glioma cells, and glioma cells inhibited the cytotoxicity against K562. Following immunomagnetic separation, the IL2 activated cells demonstrated cytotoxicity against glioma cells, K562 cells, and PHA blasts in both the CD8⁺ and the CD8^– subsets.     

腺病毒介导IL-24基因诱导人脑胶质瘤U87MG细胞的凋亡

目的：研究腺病毒介导IL-24基因表达载体(Ad-IL-24)对人脑胶质瘤U87MG细胞的抑制作用,初步探讨其作用机制。方法：将本科室构建的Ad-IL-24感染U87MG细胞,RT-PCR法检测IL-24基因的表达,MTT法和流式细胞术检测U87MG细胞的生长和凋亡,激光共聚焦显微镜观察U87MG细胞凋亡的形态学变化,RT-PCR法检测Bax、Bcl-2基因mRNA的表达,Western blotting检测caspase-3的活化。结果：Ad-IL-24感染U87MG细胞后,IL-24在U87MG细胞中有明显表达,并抑制U87MG细胞生长、诱导其凋亡、出现典型的凋亡细胞核形态学改变。Ad-IL-24可上调U87MG细胞中Bax基因、下调Bcl-2基因的表达,并诱导caspase-3蛋白的活化。结论：Ad-IL-24可诱导U87MG细胞凋亡,其机制可能与上调Bax基因、下调Bcl-2基因表达,并活化caspase-3有关。     

Adenovirus-mediated transfer of Fas ligand gene augments radiation-induced apoptosis in U-373MG glioma cells.

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.     

环状RNA在胶质瘤中的研究进展

恶性胶质瘤是中枢神经系统常见的肿瘤之一,恶性度高、难以安全切除,即使进行手术联合放化疗的综合治疗,仍有90％患者复发.目前胶质瘤的发病机制仍不明确.其在早期诊断以及长期有效的治疗上仍然面临着严峻的挑战.近年来非编码RNA备受关注.环状RNA(circular RNA,circRNA)的发现,极大地补充了传统基因表达理论...     

咖啡因通过FAK/AKT/ROCK通路调控人脑胶质瘤U-373MG细胞的恶 性生物学行为

目的：探讨咖啡因通过调节FAK/AKT/ROCK信号通路来影响人脑胶质瘤U-373MG细胞的增殖、迁移和侵袭能力。方法：常规培养U-373MG细胞,将其分为对照组、咖啡因低剂量（1 mmol/L）组、咖啡因高剂量（2 mmol/L）组、PF573228组（FAK抑制剂,1 μmol/L）、咖啡因高剂量+SC79组（AKT激活剂,8 mg/L）。用CCK-8法、Transwell小室实验、流式细胞术和WB法分别检测各组U-373MG细胞的增殖、迁移、侵袭及凋亡,以及U-373MG细胞中p-FAK、p-AKT、p-ROCK、Ki67、MMP-9蛋白表达水平。建立U-373MG细胞裸鼠移植瘤模型,观察咖啡因对移植瘤生长的影响,WB法检测移植瘤组织中相关蛋白的表达。结果：咖啡因、PF573228可显著抑制U-373MG细胞的增殖、迁移、侵袭能力,促进U-373MG细胞凋亡,抑制p-FAK、p-AKT、p-ROCK、Ki67、MMP-9蛋白的表达（均P<0.05）,SC79则可部分逆转咖啡因对U-373MG细胞的作用（均P<0.05）。咖啡因可显著抑制移植瘤的生长及移植瘤组织中上述相关蛋白的表达（均P<0.05）。结论：咖啡因可通过抑制FAK/AKT/ROCK信号通路抑制U-373MG细胞的增殖、迁移和侵袭能力,并促进其凋亡。